Bacteria in the Order Rickettsiales are obligate intracellular organisms causing major human diseases such as epidemic typhus. Emerging disease threats such as human ehrlichiosis and anaplasmosis are also caused by organisms in this group, as well as diseases that disrupt global food production through livestock mortality and morbidity. The availability of numerous genome sequences has revealed a bacterial type 4 secretion system (T4SS) in the Rickettsiales which is required for many virulence attributes of bacterial pathogens; modification of T4SS proteins often results in attenuation, providing information that could be used for vaccine development. There are significant differences between the T4SS of the Rickettsiales and the prototypical T4SS of Agrobacterium tumefaciens. These include variable, missing or extensively duplicated T4SS components and the location of the encoding genes in clusters in split genomic loci. Despite these differences, little information is available on the protein structures, interactions and topology of the T4SS in the Rickettsiales. This lack of knowledge of T4SS protein structure represents a significant gap in knowledge because of its unusual gene structure and variability, the importance of the T4SS in virulence across taxa, and the human and animal disease burden caused by the Rickettsiales. We propose here to study VirB6 of the Rickettsiales organism Anaplasma phagocytophilum. The prototypical VirB6 is an integral component of the inner membrane channel with direct substrate contact that delivers effector molecules to periplasmic and outer membrane T4SS components. In A. phagocytophilum there are four genes encoding VirB6 paralogs. VirB6-4 is an example of an extended VirB6. The VirB6-4 gene encodes a protein that is among the largest known for a T4SS, comprised of >5000 amino acids with highly repeated sequences at the C-terminus, and which varies extensively between strains. The specific aims are to identify VirB6- containing structures and interacting proteins as A. phagocytophilum invades, and investigate the microscopic organization of VirB6 during the invasion process. This investigation will provide currently lacking macromolecular structure data on the unique Rickettsiales T4SS arm of the T4SS-substrate interaction during host cell invasion.